Molecular cloning and deoxyribonucleic acid polymorphisms in Lactobacillus acidophilus and Lactobacillus gasseri.

Lactobacillus strain ADH is a bile-resistant, bacteriocin-producing human isolate that was phenotypically classified within the Lactobacillus acidophilus group. Total DNA and phage DNA extracted from strain ADH were separately digested with BclI and ligated with BclI-digested pGK12. Following electroporation of these ligation mixtures directly into strain ADH, electrotransformants were recovered at frequencies of 1.5 x 10(3) and 2.0 x 10(4)/micrograms of pGK12 for preparations of pGK12::phage DNA and pGK12::total DNA, respectively. Among the electrotransformants screened, 6 and 22% contained passenger DNA of either phage DNA or chromosomal origin, respectively, as determined by restriction-enzyme analyses and hybridization assays. Derivatives of pGK12 containing passenger DNA of chromosomal (pTRK120) or phage (pTRK121) origin and pTRK15 (native cryptic plasmid) were evaluated for use as species-specific probes. The strain ADH-derived probes hybridized primarily to members of the B-1 and B-2 lactobacilli homology groups and demonstrated strain-specific polymorphisms within these groups. Identical hybridization patterns were established for strain ADH and Lactobacillus gasseri VPI 6033 (ATCC 19992). Identification of DNA probes and establishment of a host-vector cloning system have facilitated our efforts to characterize the Lactobacillus chromosome and to distinguish between closely related species thought to be important inhabitants of the gastrointestinal tract.     

乌贼DNA条形码的筛选与验证

目的筛选适于鉴定乌贼的DNA条形码,建立鉴定舟山常见乌贼种类的DNA条形码技术体系。方法用聚合酶链反应(polymerase chain reaction, PCR)对现有5组DNA条形码引物组合进行了筛选,设计一对新的DNA条形码引物以供筛选备用。结果现有引物与部分乌贼的DNA模板存在错配而缺乏通用性,在部分乌贼DNA的扩增受阻。本实验设计的12S rRNA基因引物可以特异性扩增乌贼的DNA,提高了乌贼鉴定的准确度。结论本研究设计的12S rRNA基因引物可以作为现有乌贼DNA条形码鉴定的补充。     

Rapid detection of polymorphism in yams (Dioscorea sp) through amplification by polymerase chain reaction and rDNA variation

Ribosomal DNA (rDNA) from rice (Oriza Sativa) was used to examine polymorphism in 12 yam (Dioscorea sp) cultivars. Restriction enzyme digests of total yam DNA was probed with the pRR217 probe containing the entire repeat unit of rDNA from rice. The polymerase chain reaction was also used to amplify genomic DNA of six of the 12 cultivars studied using random primers. The amplification patterns of D rotundata-cayenensis cv tau suggested that ‘tau’ is more closely related to D rotundata sp than it is to the D cayenensis sp. The results showed polymorphisms among the different yam cultivars.     

抗菌肽BCp12对大肠杆菌壁膜及DNA损伤的作用机制

基于活菌亲和吸附法筛选的乳源蛋白抗菌肽BCp12具有广谱的抗菌效果,但其抑菌机制尚不清楚。本研究利用紫外吸收光谱、流式细胞仪、傅里叶变换红外光谱、扫描电子显微镜及透射电子显微镜等技术探究抗菌肽BCp12对大肠杆菌壁膜的损伤作用,并通过凝胶阻滞和荧光光谱实验研究BCp12与菌体DNA的结合作用及方式。结果表明：质量浓度2 mg/mL的BCp12可引起大肠杆菌壁膜亲水性增加,菌体细胞吸附率下降至64.73%,对菌体壁膜脂肪酸、蛋白、多肽酰胺、多糖及指纹信息区都有明显影响,菌体细胞膜损伤显著（损伤率为25.1%）,且细胞内紫外吸收物质泄漏,菌体形态变得粗糙皱缩,细胞质内部结构遭到严重破坏并出现空化现象;BCp12能够与溴化乙锭相互竞争结合位点,并以嵌入方式结合DNA,产生凝胶阻滞现象,影响DNA的正常复制,进而抑制菌体的生长繁殖。本研究部分揭示了BCp12的抗菌机理。     

西青果多酚对甲基苯丙胺诱导PC12细胞损伤的保护作用及机制

目的:研究西青果多酚对甲基苯丙胺(Methamphetamine,METH)诱导大鼠肾上腺嗜铬细胞瘤(PC12)细胞损伤的保护作用及机制。方法:实验分为对照组、模型组(3 mmol/L METH)和不同浓度西青果多酚组(25、50、100、200 μg/mL西青果多酚+3 mmol/L METH),给药处理24 h后检测细胞存活率、细胞凋亡、细胞DNA损伤、超氧化物歧化酶(Superoxide dismutase,SOD)和谷胱甘肽过氧化物酶(Glutathione peroxidase,GSH-Px)活力以及活性氧(Reactive oxygen species,ROS)和丙二醛(Malondialdehyde,MDA)含量等。结果:与对照组相比,3 mmol/L METH能极显著降低PC12细胞存活率(P<0.01),显著降低SOD和GSH-Px活力(P<0.05),显著增加胞内MDA含量(P<0.05),极显著增加细胞凋亡率、ROS含量和DNA损伤(P<0.01);与模型组相比,50~200 μg/mL西青果多酚能极显著抑制METH诱导的PC12细胞存活率和SOD活力的降低(P<0.01),显著抑制GSH-Px活力下降(P<0.05),极显著抑制METH导致的细胞凋亡、DNA损伤、胞内MDA和ROS含量增加(P<0.01)。结论:西青果多酚对METH诱导的PC12细胞损伤具有明显的保护作用,其作用机制可能与西青果多酚抑制METH诱导的氧化应激,缓解DNA损伤相关。     

抗高血压肽在大肠杆菌中表达的研究

本研究根据已经公开发表的具有降血压作用的来源于酪蛋白的一段多肽氨基酸序列CEI12,人工合成了一段双链DNA分子,经酶切后与表达载体pQE16连接,转化进入到大肠杆菌E.ColiJM109和E.ColiDH5嶂校又猩秆〕鲅粜灾刈榭寺。⒔杏盏急泶铮璖DS-PAGE电泳分析和westernblotting蛋白质印迹检测表明,该抗高血压小肽CEI12(与DHFR融合后)在E.ColiJM109中得到了成功表达。     

乳源抗菌肽BCp12对金黄色葡萄球菌多靶点抑菌机制

为研究新型酪蛋白源抗菌肽BCp12对金黄色葡萄球菌的抑菌机理,本实验通过酶标仪、流式细胞仪、透射电子显微镜分析BCp12对金黄色葡萄球菌的壁膜损伤机制;采用荧光光谱、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳研究BCp12对菌体DNA结合及蛋白质合成的影响;利用赖氨酸乙酰化、琥珀酰化、2-羟基异丁酰化、丙二酰化4 种泛抗体结合免疫印迹分析BCp12对菌体蛋白翻译后修饰的影响。结果表明：BCp12的最小抑菌质量浓度（minimum inhibitory concentration,MIC）为2 mg/mL,经质量浓度超过MIC的BCp12处理后的菌体细胞膜疏水性显著下降（P≤0.001）,通透性增加,菌体形变严重,部分细胞内容物外泄形成空腔;BCp12与溴化乙锭竞争性结合菌体DNA,使核酸合成受到抑制,胞内蛋白质量浓度显著下降（P≤0.001）,特别是分子质量为15～35 kDa的蛋白变化最为明显,说明BCp12可抑制菌体蛋白质的合成;金黄色葡萄球菌蛋白存在大量的赖氨酸乙酰化、琥珀酰化、丙二酰化修饰,经BCp12处理的菌体赖氨酸丙二酰化修饰水平明显下调,而对赖氨酸琥珀酰化和2-羟基异丁酰化修饰的影响不明显。本实验揭示了BCp12对金黄色葡萄球菌的多靶点抑菌机制,对新型乳源抗菌肽的应用和保障食品安全具有一定的意义。     

Novel extraction method of genomic DNA suitable for long-fragment amplification from small amounts of milk

Isolation of genomic DNA is a prerequisite for assessment of milk quality. As a source of genomic DNA, milk somatic cells from milking ruminants are practical, animal friendly, and cost-effective sources. Extracting DNA from milk can avoid the stress response caused by blood and tissue sampling of cows. In this study, we optimized a novel DNA extraction method for amplifying long (>1,000 bp) DNA fragments and used it to evaluate the isolation of DNA from small amounts of milk. The techniques used for the separation of milk somatic cell were explored and combined with a sodium dodecyl sulfate (SDS)-phenol method for optimizing DNA extraction from milk. Spectrophotometry was used to determine the concentration and purity of the extracted DNA. Gel electrophoresis and DNA amplification technologies were used for to determine DNA size and quality. The DNA of 112 cows was obtained from milk (samples of 13 ± 1 mL) and the corresponding optical density ratios at 260:280 nm were between 1.65 and 1.75. Concentrations were between 12 and 45 μg/μL and DNA size and quality were acceptable. The specific PCR amplification of 1,019- and 729-bp bovine DNA fragments was successfully carried out. This novel method can be used as a practical, fast, and economical mean for long genomic DNA extraction from a small amount of milk.     

Investigations on genetically modified maize (Bt-maize) in pig nutrition: fate of feed-ingested foreign DNA in pig bodies

The passage and fate of ingested DNA in 48 pigs fed with diets containing (n=12) parental or (n=36) transgenic (Bt) maize were examined. Pigs were fattened from an initial live weight of 24 kg to approximately 108 kg. Animals fed transgenic maize were slaughtered in groups (n=6) 4, 8, 12, 24, 48 and 72 h after feeding the last maize-containing diet. Those slaughtered at up to 12 h received no further feed, while those held for longer prior to slaughter received a diet in which maize was replaced by barley and wheat. Control animals were slaughtered at 4 and 8 h. DNA extracted from tissues and gut contents was examined by PCR for the presence of plant DNA and for any transgenic material. Recombinant DNA was detectable in the intestinal contents up to 48 h after the last feeding of a diet containing the transgenic maize. PCR amplification of plant gene spacers produced fragments of different sizes, dependent on feed source. The feed source of rectum samples depended on individual passage rate in the groups and their restriction analysis showed grain species-specific patterns. Recombinant or maize-specific DNA was not detectable in tissue samples of pigs. In contrast, plant DNA fragments were detectable in the investigated pig tissues.     

Qualitative and quantitative identification of adulteration of milk powder using DNA extracted with a novel method

The extraction of high-quality DNA from processed dairy products is often the crucial step in an authentication process by PCR-based methods. In this study, we optimized a novel DNA extraction method for milk powder and used the extracted DNA for identification of milk powder based on PCR analysis. The DNA quality was assessed by amplifying target sequences from mitochondrial genes, as well as by monitoring the yield, purity, and integrity of the extracted DNA. In addition, a laboratory adulteration model of milk powder was detected by PCR-based methods (PCR and real-time PCR) using primers targeting the mitochondrial 12S rRNA gene. Results showed that a sufficient amount and quality of DNA could be isolated from milk powder with this method. Both PCR and real-time PCR detection of cow milk compositions in goat milk powder further confirmed the DNA extracted with this extraction method could be widely used in addressing milk powder adulterant by a PCR-based method.     

基于内转录间隔区序列对云南12株鹅膏菌的分子分析

目的 基于内转录间隔区(internal transcribed spacer, ITS)序列对云南省12株鹅膏菌进行分类鉴定。方法 采用十六烷基三甲基溴化铵法(cetyltrimethylammonium Ammonium Bromide, CTAB)法提取12株鹅膏菌DNA, 以ITS4和ITS5为引物进行PCR扩增, 完成DNA序列测序, 对序列进行分析并对发育树进行构建。 结果 12个样品的ITS序列长度574~755 bp, GC含量39%~47%, 平均遗传距离为0.329, YN05、YN03与其他10株鹅膏亲缘关系较远。结论 ITS序列高度保守, 在真菌的科属种上能够初步实现对物种的鉴定和系统 发育分析, 为建立云南省鹅膏属分子数据库提供基础数据。     

Design of PCR primers and gene probes for the general detection of bacterial populations capable of degrading aromatic compounds via catechol cleavage pathways

For the general detection of bacterial populations capable of degrading aromatic compounds, two PCR primer sets were designed which can, respectively, amplify specific fragments from a wide variety of catechol 1,2-dioxygenase (C12O) and catechol 2,3-dioxygenase (C23O) genes. The C12O-targeting primer set (C12O primers) was designed based on the homologous regions of 11 C12O genes listed in the GenBank, while the C23O-targeting one (C23O primers) was designed based on those of 17 known C23O genes. Oligonucleotide probes (C12Op and C23Op) were also designed from the internal homologous regions to identify the amplified fragments. The specificity of the primer sets and probes was confirmed using authentic bacterial strains known to carry the C12O and/or C23O genes used for the primer and probe design. Various authentic bacterial strains carrying neither C12O nor C23O genes were used as negative controls. PCR with the C12O primers amplified DNA fragments of the expected sizes from 5 of the 6 known C12O-carrying bacterial strains tested, and positive signals were obtained from 4 of the 5 amplified fragments on Southern hybridization with the C12Op. The C23O primers amplified DNA fragments of the expected size from all the 11 tested C23O-carrying bacterial strains used for their design, while the C23Op detected positive signals in the amplified fragments from 9 strains. On the other hand, no DNA fragments were amplified from the negative controls. To evaluate the applicability of the designed primers and probes for the general detection of aromatic compound-degrading bacteria, they were applied to wild-type phenol- and/or benzoate-degrading bacteria newly isolated from a variety of environments. The C12O and/or C23O primers amplified DNA fragments of the expected sizes from 69 of the 106 wild-type strains tested, while the C12Op and/or C23Op detected positive signals in the amplified fragments from 63 strains. These results suggest that our primer and probe systems can detect a considerable proportion of bacteria which can degrade aromatic compounds via catechol cleavage pathways.     

Cloning of the bile salt hydrolase (bsh) gene from Enterococcus faecium FAIR-E 345 and chromosomal location of bsh genes in food enterococci

Enterococcus faecium strain FAIR-E 345 isolated from food was shown to possess bile salt hydrolase (Bsh) activity in a plate screening assay and by high-performance liquid chromatography analysis. The bsh gene was cloned and sequenced. DNA sequence analysis revealed that it encoded a protein of 324 amino acids, with pI 4.877. A bsh gene probe was prepared from the cloned bsh gene and was used for probing plasmid and total genomic DNA of Bsh-positive enterococci isolated from food to determine the genomic location of their bsh genes. This probe was able to detect the bsh gene among total genomic DNA preparations but not from plasmid preparations of 10 plasmid-bearing Enterococcus strains. However, the probe could detect the bsh gene from total genomic DNA preparations of 12 Enterococcus strains that did not contain detectable plasmid DNA. In no cases did the probe hybridize with plasmid DNA preparations, suggesting that the bsh gene among enterococci is probably generally chromosomally encoded. This presumptive chromosomal location of bsh genes among food enterococci suggests that transfer of this trait by conjugative plasmids is unlikely.     

Effect of DNA from salmon milt on human skin conditions

We investigated the effect of rubbing the DNA‐Na from salmon milt on human skin condition. A 3% DNA‐Na cream or the control cream was rubbed onto the male subjects (ten/group) for 12 weeks. The results showed that the DNA cream improved skin‐elasticity more than the control cream after the fourth week. The change in skin‐elasticity was improved significantly. Rubbing of the DNA cream tended to decrease transepithelial water loss (TEWL) and to increase water content in the face skin at 12 weeks. In addition, according to the results of a questionnaire, more than 90% of the subjects who used the DNA cream felt improving roughness of the skin. In conclusion, these results showed that their skin conditions were improved by rubbing on the DNA‐Na. We evaluated the promoting effect of the DNA‐Na on the production of hyaluronic acid using human normal dermal fibroblasts. The results showed that the DNA‐Na has the promoting effect of hyaluronic acid production on the fibroblasts. This data suggested that the DNA‐Na would improve skin conditions.     

4个引进甜菜品种DNA指纹图谱的建立

利用ISSR标记技术,对4个引进的甜菜品种建立DNA指纹图谱。结果显示,用筛选出的1条引物的PCR扩增指纹图谱即可将这4个品种区分,该引物共扩增出12个清晰条带,其中6个为共有带,多态性为50％,4个品种分别有自己的特征带。研究结果表明,利用ISSR标记指纹能够从DNA水平上明确区分甜菜不同品种,为甜菜种质鉴定提供了一种有效方法。     

Comparison of DNA quality in raw and reconstituted milk during sterilization

Responses to milk sterilization are usually evaluated only in terms of physicochemical properties and microbial safety, thus undervaluing the importance of DNA quality in an authentication process by methods based on PCR. Because DNA is a heat-sensitive molecule, we hypothesized that the heating process may impair the detection or quantification of DNA in raw fresh milk (FM) or reconstituted milk (RM), and that differences in DNA quality might exist between FM and RM. We thus investigated the effects of sterilization on the quality of DNA extracted from FM or RM; differences in DNA quality between FM and RM were also evaluated. The quality of extracted DNA from FM or RM was assessed by the specific detection of FM or RM composition in goat milk mixtures using primers targeting the bovine 12S gene, as well as by monitoring DNA yield, purity, ratio of mitochondrial (mt) to nuclear (n) DNA, and the level of DNA degradation. Polymerase chain reaction readily detected both untreated and heat-treated FM or RM in cow-goat milk mixtures, and gave a good sensitivity threshold (0.1%) under all sterilization conditions. The DNA yield and mtDNA:nDNA ratio of FM and RM varied significantly during the sterilization process. These results demonstrated that the sterilization altered the quantification of DNA in FM or RM during sterilization, but DNA could still be readily detected in sterilized FM or RM by PCR. Furthermore, we noted significant differences in DNA integrity, yield, and mtDNA:nDNA ratio between FM and RM during sterilization, which may have potential as a means to distinguish FM and RM.     

TaqMan real-time PCR for the detection and quantitation of pork in meat mixtures

A rapid and highly specific real-time quantitative PCR, based on the amplification of a fragment of the mitochondrial 12S ribosomal RNA gene (rRNA), has been developed for the quantitation of pork (Sus scrofa) in binary pork/beef muscle mixtures. The method combines the use of pork-specific primers, that amplify a 411bp fragment from pork DNA, and mammalian-specific primers amplifying a 425-428bp fragment from mammalian species DNA, which are used as endogenous control. An internal fluorogenic probe (TaqMan), that hybridizes in the "pork-specific" and also in the "mammalian" DNA fragments is used to monitor the amplification of the target gene. A comparison of the cycle number (C(t)) at which mammalian and pork-specific PCR products are first detected, in combination with the use of reference standards of known pork content, allows the determination of the percentage of pork in a mixed sample. Analysis of experimental pork/beef muscle binary mixtures demonstrated the specificity and sensitivity of the assay for detection and quantitation of pork in the range 0.5-5%.     

SNP标记用于猪肉产品DNA溯源

采用基于个体基因组DNA序列的差异而进行个体识别的DNA溯源技术,建立DNA溯源系统对肉产品的质量安全进行控制。在实验群体中(10个品种,233个个体)检测了33个新单核苷酸多态性(single nucleotide polymorphism,SNP)标记的遗传多样性,通过杂合度计算筛选出6个SNP标记可用于猪肉产品DNA溯源。进一步在屠宰场采样进行溯源模拟实验,结果表明筛选的18个SNP标记(6个新SNP标记结合已有的12个SNP标记)能有效区分100头猪个体,随机抽取的10个个体的组织样品都能通过基因型比对找到对应的个体。本研究可为早日建立猪肉产品的DNA溯源系统提供一定的技术参考。     

Detection of pea in food by real-time polymerase chain reaction (PCR)

A qualitative 5′-nuclease real-time PCR-based method for the detection of pea (Pisum sativum) in food is described. The method consists of DNA isolation by chaotropic solid phase extraction and the subsequent PCR with pea-specific primers and a TaqMan fluorescent probe. The primers and the probe are oriented to the chloroplast DNA intron located between trnL and trnF exons encoding for tRNA. The analytical parameters of the method were inclusivity 100%, exclusivity 100% and the detection limit of 0.11±0.07 ng of pea DNA corresponding to 12±7 diploid pea genome copies. Using a set of model meat patés with defined pea contents, a matrix-related detection limit of 0.05% was determined and a linear calibration line was constructed. The presented analytical method was useful for qualitative detection or semiquantitative determination of pea in food products. The method was relatively fast because the analysis could be performed in one working day.